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Kazuya Nakagomi

Publications and source records attributed to Kazuya Nakagomi.

15 recordsLinked to original sources
PubMed2006

Simple and rapid determination of hydrogen peroxide using phosphine-based fluorescent reagents with sodium tungstate dihydrate.

A simple batch method for the fluorometric determination of hydrogen peroxide using phosphine-based fluorescent reagents has been developed. A rapid, mild and selective derivatization reaction was achieved by adding sodium tungstate dihydrate to the reaction mixture of hydrogen peroxide and a phosphine-based fluorescent reagent. When 4-diphenylphosphino-7-methylthio-2,1,3-benzoxadiazole was used as a reagent, the derivatization reaction was completed after 2 min at room temperature. The calibration curve was linear between 12.5 and 500 ng hydrogen peroxide in a 10 microL sample solution. This method is accurate and has potential for on-line applications.

Calibration↗
PubMed2005

Analysis of urinary calculi obtained from a patient with idiopathic hypouricemia using micro area x-ray diffractometry and LC-MS.

Urolithiasis is a common complication in patients with hypouricemia. Using a microarea x-ray diffractometer and nanoflow liquid chromatography-mass spectrometry (LC-MS) following SDS-polyacrylamide gel electrophoresis (PAGE), recurrent urinary calculi complicating a hypouricemic patient were analyzed. Analysis with the microarea x-ray diffractometer showed that one of the calculi was composed of calcium oxalate monohydrate and hydroxyapatite. The other was found to be formed from calcium oxalate dihydrate. After determination with LC-MS, both were found to contain uromodulin, albumin, osteopontin, protein Z, and defensins. Lysozyme and calgranulin A were also identified in these calculi. Defensins, which were antimicrobial peptides, and lysozyme, a mucopeptide glycohydrolase, were identified as new organic components of urinary stones. The role of these proteins in the process of urolithiasis is of particular interest.

Amino Acid Sequence↗
PubMed2005

Chiral ligand exchange micellar electrokinetic chromatography using borate anion as a central ion.

Three compounds having 1,2-diol structure (1-phenyl-1,2-ethanediol, 3-phenoxy-1,2-propanediol, and 3-benzyloxy-1,2-propanediol) were enantioseparated by ligand exchange MEKC using (5S)-pinanediol (SPD) as a chiral selector and borate anion as a central ion together with SDS. When (S)-1,2-propanediol, (S)-1,2,4-butanetriol, or (S)-3-tert-butylamino-1,2-propanediol were used as the chiral ligand instead of SPD, these three compounds were not enantioseparated. When borate was replaced with 2-aminoethane-1-sulfonate or N-cyclohexyl-3-aminopropanesulfonate, no chiral separation was achieved. Therefore, the hydrophobic interaction between the chiral selector and the chiral analytes within the transient diastereomeric complex may play an important role in the enantioseparation achieved by the proposed method.

Borates↗
PubMed2004

Detection of protein Z in a renal calculus composed of calcium oxalate monohydrate with the use of liquid chromatography-mass spectrometry/mass spectrometry following two-dimensional polyacrylamide gel electrophoresis separation.

Protein Z, a vitamin K-dependent plasma protein, has been detected for the first time in a renal calculus along with osteopontin and prothrombin. The renal calculus was obtained from a hyperuricemic patient. Following two-dimensional polyacrylamide gel electrophoresis, the calculus was analyzed with the use of liquid chromatography mass spectrometry (LC-MS). The spectrometer was equipped with a nanoelectrospray interface and an ion trap. Four peptides were determined from a protein in the calculus through LC-MS/MS analysis. Tandem mass spectrum database matching tools were used to identify the protein as protein Z. Authentic protein Z was also analyzed using the same method, and all four peptides determined in the calculus were similarly identified. Whereas protein Z has been reported to be one of the vitamin K-dependent calcium-binding proteins, its role has not been well established. The fact that protein Z exists in a renal calculus composed of calcium oxalate will be beneficial in any future investigations into its role in the body.

Blood Proteins↗
PubMed2003

Quantification of the isomerization of Asp residue in recombinant human alpha A-crystallin by reversed-phase HPLC.

A method for determining the isomerization of Asp residues in proteins is described and demonstrated by quantifying the isomerization of Asp(151) in recombinant human alphaA-crystallin. First, four types of dodecapeptide fragment ((146)IQTGLD(151)ATHAER(157)) in which the Asp residue was either L-Asp, D-Asp, L-isoAsp or D-isoAsp were synthesized, and RP-HPLC conditions were established for their separation. Next, the Asp(151)-containing peptide fragments isolated from the tryptic hydrolysate of recombinant alphaA-crystallin were analyzed under these conditions. New peaks, the retention times of which were the same as those of peptides containing D-Asp, L-isoAsp and D-isoAsp, were generated when alphaA-crystallin was incubated for 140 days at 37 degrees C. An amino acid composition, amino acid sequence, and enantiomeric analysis revealed that two peaks with retention times identical to those of peptides containing L-isoAsp and D-isoAsp represented dodecapeptide fragments containing L-isoAsp(151) and D-isoAsp(151), respectively. RP-HPLC analysis under other condition suggested that the peak with retention time identical to that of peptide containing D-Asp represented dodecapeptide fragments containing D-Asp(151). The present method does not require acid hydrolysis, which generates further isomerization products as artifacts, and thus make possible the sensitive quantification of each type of Asp isomer individually at a specific site in a protein. In our analysis of the Asp(151) residue in human alphaA-crystallin, the degree of isomerization from L-Asp to D-Asp can be determined to a level as low as 0.3%.

Aspartic Acid↗
PubMed2003

Fluorometric determination of khellin in human urine and serum by high-performance liquid chromatography using postcolumn photoirradiation.

For the determination of khellin in urine and serum, fluorometry using HPLC-postcolumn photoirradiation has been developed. Khellin and visnagin of similar structure were separated on a column of Capcell Pak C8. The mobile phase consisted of 40%(v/v) ethanol containing 75 mmol l(-1) H2O2. The postcolumn reagent, 70 mmol l(-1) KH2PO4-NaOH buffer (pH 12.7) containing 50%(v/v) ethanol, were mixed with the mobile phase, which was irradiated with ultraviolet light to induce fluorescence. The fluorescence was monitored with excitation at 378 nm and emission at 480 nm. The calibration graph for khellin was linear over the range of 65 - 2620 ng ml(-1) using an injection volume of 20 microl. The pretreatment of the urine or serum samples consisted of diluting steps or deproteinizing steps using perchloric acid, respectively.

Calibration↗
PubMed2002

Protein domain of chicken alpha(1)-acid glycoprotein is responsible for chiral recognition.

Ovoglycoprotein from chicken egg whites (OGCHI) has been used as a chiral selector to separate drug enantiomers. However, neither the amino acid sequence of OGCHI nor the responsible part for the chiral recognition (protein domain or sugar moiety) has yet to be determined. First, we isolated a cDNA clone encoding OGCHI, and clarified the amino acid sequence of OGCHI, which consists of 203 amino acids including a predictable signal peptide of 20 amino acids. The mature OGCHI shows 31-32% identities to rabbit and human alpha(1)-acid glycoproteins (alpha(1)-AGPs). Thus, OGCHI should be the chicken alpha(1)-AGP. Second, the recombinant chicken alpha(1)-AGP was prepared by the Escherichia coli expression system, and its chiral recognition ability was confirmed by capillary electrophoresis. Since proteins expressed in E. coli are not modified by any sugar moieties, this result shows that the protein domain of the chicken alpha(1)-AGP is responsible for the chiral recognition.

Amino Acid Sequence↗
PubMed2002

Behaviors of D- and L-lactic acids during the brewing process of sake (Japanese rice wine).

The amounts of D- and L-lactic acids during the brewing process of sake were determined by capillary electrophoresis using 2-hydroxypropyl-beta-cyclodextrin as a chiral selector. Because L-lactic acid, which prevents the growth of nonuseful microorganisms, is a raw material of sake, the ratio of L-lactic acid to total lactic acid is almost 1.0 at the initial stage of sake brewing. During brewing, the ratio decreased gradually and finally reached 0.39. Yeast (Saccharomyces cerevisiae) for sake brewing produced D-lactic acid, but not L-lactic acid in a culture medium. These results suggest that the decrease in the ratio of L-lactic acid to total lactic acid during sake brewing resulted in D-lactic acid production by yeast. The ratios in 18 brands of sake obtained commercially ranged from 0.23 to 0.78. The levels of D-lactic acid in sake (140-274 mg/L) were in a narrower range than those of L-lactic acid (61-461 mg/L). Although the D-lactic acid level in sake did not correspond to total lactic acid level, the L-lactic acid level correlated well with total lactic acid level (R(2) = 0.867). These results suggest that the ratio of L-lactic acid to total lactic acid in sake reflected the amount of L-lactic acid added at the initial stage of sake brewing.

Aspergillus oryzae↗
PubMed2002

An automated fluorescence protein sequencer using 7-methylthio-4-(2,1,3-benzoxadiazolyl) isothiocyanate (MTBD-NCS) as an Edman reagent.

An automated fluorescence protein sequencer using 7-methylthio-5-(2,1,3-benzoxadiazolyl) isithiocyanate (MTBD-NCS), a fluorescent Edman reagent, is developed by the modification of a commercial protein sequencer. The generated MTBD-thiohydantoin amino acids fluoresced strongly, whereas the by-products such as MTBD-thiocarbamoyl amino acids and MTBD-carbamoly amino acids did not fluoresce. A few interfering peaks were observed in the chromatogram and amino acid sequence was easily determined. The coupling and cyclization/cleavage reaction conditions and extraction conditions of generated MTBD-thiazolinone amino acids were optimized using an autonalyzer. Finally, the sequence of a synthetic peptide (25 pmol), leucine-enkephalin-Thr-amide, was determined and up to six residues were successively analyzed.

Fluorescent Dyes↗
PubMed2002

A rapid assay method for catechol-O-methyltransferase activity by flow injection analysis.

A rapid assay employing flow injection analysis (FIA) to determine the activity of purified catechol-O-methyltransferase (COMT) from porcine liver is described. The method was based on the determination of normetanephrine, the 3-O-methyl metabolite of the substrate norepinephrine. Excess norepinephrine was removed from the incubation mixture by alumina extraction twice to allow normetanephrine to be subjected to flow injection analysis, coulometrical oxidation, fluorogenic reaction with ethylenediamine and fluorescence detection. K(m) and V(max) values for COMT obtained with the system were 503 microM and 4.51 nmol/min/mg protein, respectively. The method is suitable for screening of COMT inhibitors or activators, as a large number of samples, up to 200, can be processed in one working day.

Animals↗
PubMed2002

Isolation of cathepsin B inhibitory peptides, Cabin-A1 and -A2, from a tryptic and chymotryptic hydrolysate of human serum albumin.

Two novel peptides that inhibit cathepsin B were isolated from a tryptic and chymotryptic hydrolysate of human serum albumin, and designated as Cabin-A1 and -A2. Cabin-A1 and -A2 were purified by reversed-phase HPLC and identified as Ser-Leu-His-Thr-Leu-Phe and Phe-Gln-Asn-Ala-Leu, respectively. These peptides correspond to f(65-70) and f(403-407) of human serum albumin. Human albutensin A (Ala-Phe-Lys-Ala-Trp-Ala-Val-Ala-Arg), which corresponds to f(210-218), was also isolated as a potent cathepsin B inhibitor. Synthetic Cabin-A1, -A2, and human albutensin A showed dose-dependent inhibition of cathepsin B, with K(i) values of 2.4, 290, and 3.8 microM, respectively.

Cathepsin B↗
PubMed2002

An automated analyzer for methylated arginines in rat plasma by high-performance liquid chromatography with post-column fluorescence reaction.

A fully automated analyzer for methylated L-arginine metabolites [N,N-dimethyl-L-arginine (ADMA), N-methylarginine (NMMA) and N,N'-dimethyl-L-arginine (SDMA)] by high-performance liquid chromatography with post-column fluorescence derivatization was developed. This system consists of an on-line extraction, a separation on a reversed phase ion-pair chromatograph, a post-column derivatization by o-phthaladehyde (OPA) and thiol reaction, and fluorescence detection. NMMA, ADMA and SDMA were separated in 40 min with isocratic elution by a combination of octanoate and cyclohexane carboxylate as ion-pair reagents. The eluate was monitored at 450 nm with excitation at 337 nm. The calibration curves for NMMA, ADMA and SDMA showed linearity over the range from 0.05 micromol l(-1) (0.5 pmol on column) to 5.0 micromol l(-1) (50 pmol on column). This method does not require any time-consuming pre-treatment and requires only 10 microl of plasma sample for assay.

Animals↗
PubMed2002

Isolation of novel peptides, cabin-1, -2, -3, and -4, that inhibit cathepsin B from a thermolysin digest of human plasma.

Four novel peptides that inhibit cathepsin B, designated as Cabin-1, -2, -3, and -4, were isolated from a thermolysin digest of human plasma. After gel filtration and cation-exchange chromatography, the peptide mixture was purified by reverse-phase HPLC to isolate Cabin-1, -2, -3, and 4, with the amino acid sequences LGPVTQE, VLQSSGLYS, VVSVLT, and LVYDAY, respectively. These peptides correspond to f(64-70) of human apolipoprotein A-I for Cabin-1, f(56-64) and f(185-190) of the human immunoglobulin G gamma chain for Cabin-2 and -3, and f(66-71) of human transferrin for Cabin-4. Synthetic Cabin-1, -2, -3, and -4 showed dose-dependent inhibition of cathepsin B. Their IC50 values were 450, 500, 20, and 5.0 micromol/l, respectively. Lineweaver-Burk plots suggested that Cabin-3 is a noncompetitive inhibitor, while Cabin-4 is a competitive inhibitor. Among the N- and C-terminal deletion peptides of Cabin-2 and -4, Cabin-2(1-8), VLQSSGLY, was found to have the most potent inhibitory activity, with an IC50 of 3.8 micromol/l.

Cathepsin B↗
PubMed2002

Improvement in the properties of 3-phenyl-3-trifluoromethyldiazirine based photoreactive bis-glucose probes for GLUT4 following substitution on the phenyl ring.

We have developed two novel 3-phenyl-3-trifluoromethyldiazirinyl bis-glucose derivatives to investigate the properties of the adipocyte glucose transporter GLUT4. These compounds were substituted by electron-withdrawing (iodo and nitro) groups on the aromatic ring of 3-phenyl-3-trifluoromethyldiazirine photophore and were found to be more photosensitive than compounds without such substituents. The compounds were used as inhibitors of insulin-stimulated glucose transport activity in order to assess half-maximal inhibition or relative affinity values for GLUT4. The affinities were found to be 60-130 times higher than the parent compound D-glucose. Because of the increased photo-reactivity and high affinity these compounds will be useful in studies directed at further elucidation of GLUT4 function.

Adipocytes↗